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Abstract. Liver disease involves a complex interplay of pathological processes, including inflammation, hepatocyte necrosis, and fibrosis. End-stage liver disease (ESLD), such as liver failure and decompensated cirrhosis, has a high mortality rate, and liver transplantation is the only effective treatment. However, to overcome problems such as the shortage of donor livers and complications related to immunosuppression, there is an urgent need for new treatment strategies that need to be developed for patients with ESLD. For instance, hepatocytes derived from donor livers or stem cells can be engrafted and multiplied in the liver, substituting the host hepatocytes and rebuilding the liver parenchyma. Stem cell therapy, especially mesenchymal stem cell therapy, has been widely proved to restore liver function and alleviate liver injury in patients with severe liver disease, which has contributed to the clinical application of cell therapy. In this review, we discussed the types of cells used to treat ESLD and their therapeutic mechanisms. We also summarized the progress of clinical trials around the world and provided a perspective on cell therapy.

Abstract Exosomes can regulate the malignant progression of tumors by carrying a variety of genetic information and transmitting it to target cells. Recent studies indicate that exosomal circular RNAs (circRNAs) regulate multiple biological processes in carcinogenesis, such as tumor growth, metastasis, epithelial–mesenchymal transition, drug resistance, autophagy, metabolism, angiogenesis, and immune escape. In the tumor microenvironment (TME), exosomal circRNAs can be transferred among tumor cells, endothelial cells, cancer‐associated fibroblasts, immune cells, and microbiota, affecting tumor initiation and progression. Due to the high stability and widespread presence of exosomal circRNAs, they hold promise as biomarkers for tumor diagnosis and prognosis prediction in blood and urine. In addition, designing nanoparticles targeting exosomal circRNAs and utilizing exosomal circRNAs derived from immune cells or stem cells provide new strategies for cancer therapy. In this review, we examined the crucial role of exosomal circRNAs in regulating tumor‐related signaling pathways and summarized the transmission of exosomal circRNAs between various types of cells and their impact on the TME. Finally, our review highlights the potential of exosomal circRNAs as diagnostic and prognostic prediction biomarkers, as well as suggesting new strategies for clinical therapy.

Abstract Background Neural Tube Defects (NTDs) are congenital malformations of the central nervous system resulting from the incomplete closure of the neural tube during early embryonic development. Neuroinflammation refers to the inflammatory response in the nervous system, typically resulting from damage to neural tissue. Immune-related processes have been identified in NTDs, however, the detailed relationship and underlying mechanisms between neuroinflammation and NTDs remain largely unclear. In this study, we utilized integrated multi-omics analysis to explore the role of neuroinflammation in NTDs and identify potential prenatal diagnostic markers using a murine model. Methods Nine public datasets from Gene Expression Omnibus (GEO) and ArrayExpress were mined using integrated multi-omics analysis to characterize the molecular landscape associated with neuroinflammation in NTDs. Special attention was given to the involvement of macrophages in neuroinflammation within amniotic fluid, as well as the dynamics of macrophage polarization and their interactions with neural cells at single-cell resolution. We also used qPCR assay to validate the key TFs and candidate prenatal diagnostic genes identified through the integrated analysis in a retinoic acid-induced NTDs mouse model. Results Our analysis indicated that neuroinflammation is a critical pathological feature of NTDs, regulated both transcriptionally and epigenetically within central nervous system tissues. Key alterations in gene expression and pathways highlighted the crucial role of STATs molecules in the JAK-STAT signaling pathway in regulating NTDs-associated neuroinflammation. Furthermore, single-cell resolution analysis revealed significant polarization of macrophages and their interaction with neural cells in amniotic fluid, underscoring their central role in mediating neuroinflammation associated with NTDs. Finally, we identified a set of six potential prenatal diagnostic genes, including FABP7, CRMP1, SCG3, SLC16A10, RNASE6 and RNASE1, which were subsequently validated in a murine NTDs model, indicating their promise as prospective markers for prenatal diagnosis of NTDs. Conclusions Our study emphasizes the pivotal role of neuroinflammation in the progression of NTDs and underlines the potential of specific inflammatory and neural markers as novel prenatal diagnostic tools. These findings provide important clues for further understanding the underlying mechanisms between neuroinflammation and NTDs, and offer valuable insights for the future development of prenatal diagnostics.

Oncolytic virotherapy represents a promising approach in cancer immunotherapy. The primary delivery method for oncolytic viruses (OVs) is intratumoral injection, which apparently limits their clinical application. For patients with advanced cancer with disseminated metastasis, systemic administration is considered the optimal approach. However, the direct delivery of naked viruses through intravenous injection presents challenges, including rapid clearance by the immune system, inadequate accumulation in tumors, and significant side effects. Consequently, the development of drug delivery strategies has led to the emergence of various bio-materials serving as viral vectors, thereby improving the anti-tumor efficacy of oncolytic virotherapy. This review provides an overview of innovative strategies for delivering OVs, with a focus on nanoparticle-based or cell-based delivery systems. Recent pre-clinical and clinical studies are examined to highlight the enhanced efficacy of systemic delivery using these novel platforms. In addition, prevalent challenges in current research are briefly discussed, and potential solutions are proposed.

BackgroundMajor depressive disorder (MDD) pathogenesis may involve metalloids in a significant way. The aim of our study was to identify potential links between MDD and metalloid elements [boron (B), germanium (Ge), arsenic (As), antimony (Sb)].MethodsA total of 72 MDD cases and 75 healthy controls (HCs) were recruited from Zhumadian Second People’s Hospital in Henan Province, China. The levels of four metallic elements (B, Ge, As, and Sb) in the serum and urine were measured using inductively coupled plasma mass spectrometry (ICP-MS).ResultsIn comparison to the HCs, the B, As, and Sb levels were considerably lower in the MDD group (p < 0.05) in the serum; the MDD group had significantly higher (p < 0.05) and significantly lower (p < 0.001) B and Sb levels in the urine. After adjusting for potential confounders, serum B (OR = 0.120; 95% CI, 0.048, 0.300; p < 0.001) and Sb (OR = 0.133; 95% CI, 0.055, 0.322; p < 0.001) showed a negative correlation with MDD. Urine B had a negative correlation (OR = 0.393; 95% CI, 0.193, 0.801; p = 0.01) with MDD, while urine Sb had a positive correlation (OR = 3.335; 95% CI, 1.654, 6.726; p = 0.001) with MDD. ConclusionOur current research offers insightful hints for future investigation into the function of metalloids in connection to MDD processes.

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide. One of the main causes of colorectal cancer is inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD). Intestinal epithelial cells (IECs), intestinal mesenchymal cells (IMCs), immune cells, and gut microbiota construct the main body of the colon and maintain colon homeostasis. In the development of colitis and colitis-associated carcinogenesis, the damage, disorder or excessive recruitment of different cells such as IECs, IMCs, immune cells and intestinal microbiota play different roles during these processes. This review aims to discuss the various roles of different cells and the crosstalk of these cells in transforming intestinal inflammation to cancer, which provides new therapeutic methods for chemotherapy, targeted therapy, immunotherapy and microbial therapy.

Abstract Cancer and impaired tissue wound healing with ageing are closely related to the quality of life of the elderly population. Given the increased incidence of cancer and the population ageing trend globally, it is very important to explore how ageing impairs tissue wound healing and spontaneous cancer. In a murine model of DSS-induced acute colitis and AOM/DSS-induced colitis-associated cancer (CAC), we found ageing significantly decreases intestinal wound healing and simultaneous CAC initiation, although ageing does not affect the incidence of AOM-induced, sporadic non-inflammatory CRC. Mechanistically, reduced fibroblasts were observed in the colitis microenvironment of ageing mice. Through conditional lineage tracing, an important source of fibroblasts potentially derived from intestinal smooth muscle cells (ISMCs) was identified orchestrating intestinal wound healing and CAC initiation in young mice. However, the number of transformed fibroblasts from ISMCs significantly decreased in ageing mice, accompanied by decreased intestinal wound healing and decreased CAC initiation. ISMCs-fibroblasts transformation in young mice and reduction of this transformation in ageing mice were also confirmed by ex-vivo intestinal muscular layer culture experiments. We further found that activation of YAP/TAZ in ISMCs is required for the transformation of ISMCs into fibroblasts. Meanwhile, the reduction of YAP/TAZ activation in ISMCs during intestinal wound healing was observed in ageing mice. Conditional knockdown of YAP/TAZ in ISMCs of young mice results in reduced fibroblasts in the colitis microenvironment, decreased intestinal wound healing and decreased CAC initiation, similar to the phenotype of ageing mice. In addition, the data from intestine samples derived from inflammatory bowel disease (IBD) patients show that activation of YAP/TAZ also occurs in ISMCs from these patients. Collectively, our work reveals an important role of the ageing stromal microenvironment in intestinal wound healing and CAC initiation. Furthermore, our work also identified a potential source of fibroblasts involved in colitis and CAC.

Abstract Background Decompensated liver cirrhosis (DLC), a terminal-stage complication of liver disease, is a major cause of morbidity and mortality in patients with hepatopathies. Human umbilical cord mesenchymal stem cell (hUCMSC) therapy has emerged as a novel treatment alternative for the treatment of DLC. However, optimized therapy protocols and the associated mechanisms are not entirely understood. Methods We constructed a DLC rat model consistent with the typical clinical characteristics combined use of PB and CCL4. Performing dynamic detection of liver morphology and function in rats for 11 weeks, various disease characteristics of DLC and the therapeutic effect of hUCMSCs on DLC in experimental rats were thoroughly investigated, according to ascites examination, histopathological, and related blood biochemical analyses. Flow cytometry analysis of rat liver, immunofluorescence, and RT-qPCR was performed to examine the changes in the liver immune microenvironment after hucMSCs treatment. We performed RNA-seq analysis of liver and primary macrophages and hUCMSCs co-culture system in vitro to explore possible signaling pathways. PPARγ antagonist, GW9662, and clodronate liposomes were used to inhibit PPAR activation and pre-exhaustion of macrophages in DLC rats’ livers, respectively. Results We found that changing the two key issues, the frequency and initial phase of hUCMSCs infusion, can affect the efficacy of hUCMSCs, and the optimal hUCMSCs treatment schedule is once every week for three weeks at the early stage of DLC progression, providing the best therapeutic effect in reducing mortality and ascites, and improving liver function in DLC rats. hUCMSCs treatment skewed the macrophage phenotype from M1-type to M2-type by activating the PPARγ signaling pathway in the liver, which was approved by primary macrophages and hUCMSCs co-culture system in vitro. Both inhibition of PPARγ activation with GW9662 and pre-exhaustion of macrophages in DLC rats’ liver abolished the regulation of hUCMSCs on macrophage polarization, thus attenuating the beneficial effect of hUCMSCs treatment in DLC rats. Conclusions These data demonstrated that the optimal hUCMSCs treatment effectively inhibits the ascites formation, prolongs survival and significantly improves liver structure and function in DLC rats through the activation of the PPARγ signaling pathway within liver macrophages. Our study compared the efficacy of different hUCMSCs infusion regimens for DLC, providing new insights on cell-based therapies for regenerative medicine.

Abstract Mucopolysaccharidosis type I (MPS I) is a severe disease caused by loss-of-function mutation variants in the α-L-iduronidase (Idua) gene. In vivo genome editing represents a promising strategy to correct Idua mutations, and has the potential to permanently restore IDUA function over the lifespan of patients. Here, we used adenine base editing to directly convert A > G (TAG>TGG) in a newborn murine model harboring the Idua-W392X mutation, which recapitulates the human condition and is analogous to the highly prevalent human W402X mutation. We engineered a split-intein dual-adeno-associated virus 9 (AAV9) adenine base editor to circumvent the package size limit of AAV vectors. Intravenous injection of the AAV9-base editor system into MPS IH newborn mice led to sustained enzyme expression sufficient for correction of metabolic disease (GAGs substrate accumulation) and prevention of neurobehavioral deficits. We observed a reversion of the W392X mutation in 22.46 ± 6.74% of hepatocytes, 11.18 ± 5.25% of heart and 0.34 ± 0.12% of brain, along with decreased GAGs storage in peripheral organs (liver, spleen, lung and kidney). Collectively, these data showed the promise of a base editing approach to precisely correct a common genetic cause of MPS I in vivo and could be broadly applicable to the treatment of a wide array of monogenic diseases.

Summary: Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5–10.5 days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

Abstract Background Inflammatory bowel diseases (IBD) are chronic relapsing–remitting inflammatory diseases of the gastrointestinal tract that are typically categorized into two subtypes: Crohn's disease (CD) and ulcerative colitis (UC). Although MSCs therapy has achieved encouraging outcomes in IBD therapy, objective responses are limited in colon fibrosis stenosis owing to the complicated microenvironment of CD and MSCs heterogeneity of quality. Here, we chose IFN-γ and kynurenic acid (KYNA) to overcome the low response and heterogeneity of human adipose-derived MSCs (hADSCs) to treat IBD and expand the therapeutic effects based on the excellent ability of IFN-γ and KYNA to promote indoleamine 2,3-dioxygenase-1 (IDO-1) signaling, providing a potential protocol to treat IBD and fibrosis disease. Methods hADSCs were isolated, cultured, and identified from human abdominal adipose tissue. The CD pathology-like acute colitis and chronic colon fibrosis rat model was induced by 2,4,6-trinitrobenzen sulfonic acid (TNBS). hADSCs were pretreated in vitro with IFN-γ and KYNA and then were transplanted intravenously at day 1 and 3 of TNBS administration in colitis along with at day 1, 15, and 29 of TNBS administration in chronic colonic fibrosis. Therapeutic efficacy was evaluated by body weights, disease activity index, pathological staining, real-time PCR, Western blot, and flow cytometry. For knockout of IDO-1, hADSCs were transfected with IDO-1-targeting small gRNA carried on a CRISPR-Cas9-lentivirus vector. Results hADSCs treated with IFN-γ and KYNA significantly upregulated the expression and secretion of IDO-1, which has effectively ameliorated CD pathology-like colitis injury and fibrosis. Notably, the ability of hADSCs with IDO-1 knockout to treat colitis was significantly impaired and diminished the protective effects of the primed hADSCs with IFN-γ and KYNA. Conclusion Inflammatory cytokines IFN-γ- and KYNA-treated hADSCs more effectively alleviate TNBS-induced colitis and colonic fibrosis through an IDO-1-dependent manner. Primed hADSCs are a promising new strategy to improve the therapeutic efficacy of MSCs and worth further research.

Abstract Background The pathophysiological mechanisms of aggression are manifold and they may closely interconnect. Current study aimed to determine the gut microbiota and its metabolites, and clarify their correlations with inflammation, oxidation, leaky gut and clinical profiles underlying aggression in schizophrenia (ScZ). Methods Serum and stool specimens from ScZ inpatients with (ScZ-Ag, 25 cases) and without aggression (NScZ-Ag, 25 cases) were collected. Systemic inflammation, oxidation and leaky gut biomarkers were determined by ELISA, gut microbiota by 16S rRNA sequencing, short-chain fatty acids (SCFAs) by gas chromatography-mass spectrometry analysis and neurotransmitters by liquid chromatograph mass spectrometry analysis. Results Significantly higher systemic pro-inflammation, pro-oxidation and leaky gut biomarkers were observed in ScZ-Ag than NScZ-Ag group (all P

Glioblastoma is a malignant tumor with the highest morbidity and mortality in the central nervous system. Conventional surgical resection combined with radiotherapy or chemotherapy has a high recurrence rate and poor prognosis. The 5-year survival rate of patients is less than 10%. In tumor immunotherapy, CAR-T cell therapy represented by chimeric antigen receptor-modified T cells has achieved great success in hematological tumors. However, the application of CAR-T cells in solid tumors such as glioblastoma still faces many challenges. CAR-NK cells are another potential adoptive cell therapy strategy after CAR-T cells. Compared with CAR-T cell therapy, CAR-NK cells have similar anti-tumor effects. CAR-NK cells can also avoid some deficiencies in CAR-T cell therapy, a research hotspot in tumor immunity. This article summarizes the preclinical research status of CAR-NK cells in glioblastoma and the problems and challenges faced by CAR-NK in glioblastoma.

Mucopolysaccharidosis type I-Hurler (MPS I-H) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the α-L-iduronidase (IDUA) gene. Current treatments are ineffective for treating central nervous system (CNS) manifestations because lysosomal enzymes do not effectively cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, we engineered a modified IDUA protein by adding a brain-targeting peptide from melanotransferrin. We demonstrated that fusion of melanotransferrin peptide (MTfp) at the N terminus of human IDUA (hIDUA) was enzymatically active and could efficiently cross the BBB in vitro. Then, liver-directed gene therapy using the adeno-associated virus 8 (AAV8) vector, which encoded the modified hIDUA cDNA driven by a liver-specific expression cassette was evaluated in an adult MPS I-H mouse model. The results showed that intravenous (i.v.) infusion of AAV8 resulted in sustained supraphysiological levels of IDUA activity and normalized glycosaminoglycan (GAG) accumulation in peripheral tissues. Addition of MTfp to the hIDUA N terminus allowed efficient BBB transcytosis and IDUA activity restoration in the brain, resulting in significant improvements in brain pathology and neurobehavioral deficits. Our results provide a novel strategy to develop minimally invasive therapies for treatment of MPS I-H and other neurodegenerative LSDs.

Inhibition of vascular endothelial growth factor (VEGF) is the standard therapy for neovascular age-related macular degeneration (nAMD). However, anti-VEGF agents used in the clinic require repeated injections, causing adverse effects. Gene therapy could provide sustained anti-VEGF levels after a single injection, thereby drastically decreasing the treatment burden and improving visual outcomes. In this study, we developed a novel VEGF Trap, nVEGFi, containing domains 1 and 2 of VEGFR1 and domain 3 of VEGFR2 fused to the Fc portion of human IgG. The nVEGFi had a higher expression level than aflibercept under the same expression cassettes of adeno-associated virus (AAV)8 in vitro and in vivo. nVEGFi was found to be noninferior to aflibercept in binding and blocking VEGF in vitro. AAV8-mediated expression of nVEGFi was maintained for at least 12 weeks by subretinal delivery in C57BL/6J mice. In a mouse laser-induced choroidal neovascularization (CNV) model, 4 × 108 genome copies of AAV8-nVEGFi exhibited a significantly increased reduction in the CNV area compared with AAV8-aflibercept (78.1% vs. 63.9%, p < 0.05), while causing no structural or functional changes to the retina. In conclusion, this preclinical study showed that subretinal injection of AAV8-nVEGFi was long lasting, well tolerated, and effective for nAMD treatment, supporting future translation to the clinic.

Distant metastasis is a major cause of death in patients with colorectal cancer (CRC) but the management of advanced and metastatic CRC still remains problematic due to the distinct molecular alterations during tumor progression. Tumor angiogenesis is a key step in tumor growth, invasion and metastasis. However, the signaling pathways involved in angiogenesis are poorly understood. The results of the present study showed that secretogranin II (SCG2) was significantly downregulated in malignant CRC tissues, and higher expression of SCG2 was correlated with longer disease‐free survival and overall survival of CRC patients. The results of an animal study showed that ectopic expression of SCG2 significantly inhibited CRC tumor growth by disrupting angiogenesis. Furthermore, the inhibition of expression of vascular endothelial growth factor (VEGF) by SCG2 and rescue of VEGF effectively blocked SCG2‐induced inhibition of angiogenesis. Investigations into the underlying mechanism suggested that SCG2 promoted degradation of hypoxia‐inducible factor (HIF)‐1α by interacting with the von Hippel–Lindau tumor suppressor in CRC cells. Blocking of degradation of HIF‐1α effectively attenuated the SCG2‐mediated decrease in expression of VEGF in CRC cells. Collectively, these results demonstrated that treatment with SCG2 effectively inhibited CRC tumor growth by disrupting the activities of HIF‐1α/VEGF, thereby clarifying the anti‐tumor and anti‐angiogenesis roles of SCG2 in CRC, while providing a novel therapeutic target and a potential prognostic marker of disease progression.

Malignant ascites frequently occur in patients with advanced ovarian cancer at initial diagnosis, and in almost all cases of relapse, they are closely related to poor prognosis, chemoresistance, and metastasis. To date, effective management strategies have been limited. In this study, we aimed to investigate the effects of oncolytic adenovirus (OV) on malignant ascites in a mouse model of advanced ovarian cancer. The results suggested that OV conferred an effective ability to reduce ascites development and prolong overall survival. Further analysis of the ascitic immune microenvironment revealed that OV treatment promoted T cell infiltration, activation, and differentiation into the effector phenotype; reprogrammed macrophages toward the M1-like phenotype; and increased the ratios of both CD8+ T cells to CD4+ T cells and M1 to M2 macrophages. However, immunosuppressive factors such as PD-1, LAG-3, and Tregs emerged after treatment. Combination therapy including OV, CSF-1R inhibitor PLX3397, and anti-PD-1 remarkably delayed the progression of ascites, and combination therapy induced a greater extent of T cell infiltration, proliferation, and activation. This study provides experimental and theoretical evidence for oncolytic virus-based treatment of malignant ascites, which may further contribute to advanced ovarian cancer therapy.

A major challenge in natural killer (NK) cell immunotherapy is the limited persistence of NK cells in vivo. However, the proliferation of NK cells is dependent on cytokines such as interleukin-2 (IL-2). Although IL-2 is a critical cytokine for NK cell activation and survival, IL-2 administration in adoptive NK cell therapy can induce adverse toxicities. To improve the persistence of NK cells and attenuate the systemic toxicity of IL-2, we constructed a cell-restricted artificial IL-2, named membrane-bound IL-2 (mbIL-2), comprising human IL-2 and human IL-2Rα joined by a classic linker. We found that mbIL-2-activated NK-92 cells can survive and proliferate in vitro and in vivo, independent of exogenous IL-2, while mbIL-2-expressing NK-92 cells do not support bystander cell survival or proliferation. Additionally, mbIL-2 enhanced NK-92 cell-mediated antitumor activity by tuning the IL-2 receptor downstream signals and NK cell receptor repertoire expression. To conclude, our novel mbIL-2 improves NK-92 cell persistence and enhances NK-92 cell-mediated antitumor activity. NK-92 cells genetically modified to express the novel mbIL-2 with potential significance for clinical development.

Long noncoding RNA (lncRNA) plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). The gene mutations of adenomatous polyposis coli (APC) were found in most patients with CRC. They function as important inducers of tumorigenesis. Based on our microarray results, we identified a specific upregulated lncRNA in CRC (SURC). Further analysis showed that high SURC expression correlated with poorer disease-free survival and overall survival in patients with CRC. Furthermore, we found that mutated APC genes can promote the transcription of SURC by reducing the degradation of β-catenin protein in CRC. Functional assays revealed that knockdown of SURC impaired CRC cell proliferation, colony formation, cell cycle, and tumor growth. Additionally, SURC promotes CCND2 expression by inhibiting the expression of miR–185-5p in CRC cells. In conclusion, we demonstrate that SURC is a specific upregulated lncRNA in CRC and the SURC/miR–185-5p/CCND2 axis may be targetable for CRC diagnosis and therapy.

Abstract Malignant transformation of gastrointestinal stromal tumors (GISTs) is correlated with poor prognosis; however, the underlying biological mechanism is not well understood. In the present study, low-risk (LR) GISTs, GISTs categorized as high-risk based on tumor size (HBS), and on mitotic rate (HBM) were collected for RNA sequencing. Candidate hub lncRNAs were selected by Oncomine analysis. Expression of a selected hub lncRNA, DNM3OS, and its correlation with patients’ prognosis were analyzed using FISH staining, followed with the determination of function and underlying mechanism. Our results revealed a series of key pathways and hub lncRNAs involved in the malignant transformation of GISTs. Oncomine analysis revealed a tight association between clinical signatures and DNM3OS and suggested that DNM3OS is a hub lncRNA that is involved in the Hippo signaling pathway. In addition, DNM3OS was upregulated in HBS, HBM, and HBS/M GIST and correlated with worse prognosis in patients with GISTs. In addition, DNM3OS promoted GIST cell proliferation and mitosis by regulating the expression of GLUT4 and CD36. Collectively, these results improve our understanding of the malignant transformation of GISTs and unveil a series of hub lncRNAs in GISTs.

Abstract Background Direct reprogramming of human fibroblasts to hepatocyte-like cells was proposed to generate large-scale functional hepatocytes demanded by liver tissue engineering. However, the difficulty in obtaining large quantities of human fibroblasts greatly restricted the extensive implementation of this approach. Meanwhile, human umbilical cord mesenchymal stem cells (HUMSCs) are the preferred cell source for HLCs with the advantages of limited ethical concerns, easy accessibility, and propagation in vitro. However, no direct reprogramming protocol for converting HUMSCs to hepatoblast-like cells (HLCs) has been reported. Methods HLCs were successfully generated from HUMSCs by forced expression of FOXA3, HNF1A, and HNF4A (collectively as 3TFs) and c-Myc. In vitro and in vivo functional experiments were conducted to demonstrate the hepatic phenotype, characterization, and function of HUMSC-derived HLCs (HUMSC-iHeps). ChIP-seq and RNA-seq were integrated to reveal the potential molecular mechanisms underlying c-Myc-mediated reprogramming. Results We showed that c-Myc greatly improved the trans-differentiation efficiency for HLCs from HUMSCs, which remained highly efficient in reprogramming fibroblasts into HLCs, suggesting c-Myc could promote direct reprogramming and its potentially widespread applicability for generating large amounts of HLCs in vitro. Mice transplantation experiments further confirmed the therapeutic potential of HUMSC-iHeps by liver function restoration and survival prolongation. Besides, in vivo safety assessment demonstrated the low risk of the tumorigenic potential of HUMSC-iHeps. We found that c-Myc functioned predominantly at an early phase of reprogramming, and we further unraveled the regulatory network altered by c-Myc. Conclusions c-Myc enhanced reprogramming efficiency of HLCs from HUMSCs. A large scale of functional HLCs generated more conveniently from HUMSCs could benefit biomedical studies and applications of liver diseases.

Abstract Mesenchymal stem cell (MSC)-mediated immunomodulation has been harnessed for the treatment of human diseases, but its underlying mechanism has not been fully understood. Dead cells, including apoptotic cells have immunomodulatory properties. It has been repeatedly reported that the proportion of nonviable MSCs in a MSC therapeutic preparation varied from 5~50% in the ongoing clinical trials. It is conceivable that the nonviable cells in a MSC therapeutic preparation may play a role in the therapeutic effects of MSCs. We found that the MSC therapeutic preparation in the present study had about 5% dead MSCs (DMSCs), characterized by apoptotic cells. Namely, 1 × 106 MSCs in the preparation contained about 5 × 104 DMSCs. We found that the treatment with even 5 × 104 DMSCs alone had the equal therapeutic effects as with 1 × 106 MSCs. This protective effect of the dead MSCs alone was confirmed in four mouse models, including concanavalin A (ConA)- and carbon tetrachloride (CCl4)-induced acute liver injury, LPS-induced lung injury and spinal cord injury. We also found that the infused MSCs died by apoptosis in vivo. Furthermore, the therapeutic effect was attributed to the elevated level of phosphatidylserine (PS) upon the injection of MSCs or DMSCs. The direct administration of PS liposomes (PSLs) mimic apoptotic cell fragments also exerted the protective effects as MSCs and DMSCs. The Mer tyrosine kinase (MerTK) deficiency or the knockout of chemokine receptor C–C motif chemokine receptor 2 (CCR2) reversed these protective effects of MSCs or DMSCs. These results revealed that DMSCs alone in the therapeutic stem cell preparation or the apoptotic cells induced in vivo may exert the same immunomodulatory property as the “living MSCs preparation” through releasing PS, which was further recognized by MerTK and participated in modulating immune cells.

Abstract Background Liver fibrosis (LF) is a common pathological process characterized by the activation of hepatic stellate cells (HSCs) and accumulation of extracellular matrix. Severe LF causes cirrhosis and even liver failure, a major cause of morbidity and mortality worldwide. Transplantation of human placental mesenchymal stem cells (hPMSCs) has been considered as an alternative therapy. However, the underlying mechanisms and the appropriate time window for hPMSC transplantation are not well understood. Methods We established mouse models of CCl4-injured LF and administered hPMSCs at different stages of LF once a week for 2 weeks. The therapeutic effect of hPMSCs on LF was investigated, according to histopathological and blood biochemical analyses. In vitro, the effect of hPMSCs and the secretomes of hPMSCs on the inhibition of activated HSCs was assessed. RNA sequencing (RNA-seq) analysis, real-time PCR array, and western blot were performed to explore possible signaling pathways involved in treatment of LF with hPMSCs. Results hPMSC treatment notably alleviates experimental hepatic fibrosis, restores liver function, and inhibits inflammation. Furthermore, the therapeutic effect of hPMSCs against mild-to-moderate LF was significantly greater than against severe LF. In vitro, we observed that the hPMSCs as well as the secretomes of hPMSCs were able to decrease the activation of HSCs. Mechanistic dissection studies showed that hPMSC treatment downregulated the expression of fibrosis-related genes, and this was accompanied by the upregulation of Caveolin-1 (Cav1) (p < 0.001). This suggested that the amelioration of LF occurred partly due to the restoration of Cav1 expression in activated HSCs. Upregulation of Cav1 can inhibit the TGF-β/Smad signaling pathway, mainly by reducing Smad2 phosphorylation, resulting in the inhibition of activated HSCs, whereas this effect could be abated if Cav1 was silenced in advance by siRNAs. Conclusions Our findings suggest that hPMSCs could provide multifaceted therapeutic benefits for the treatment of LF, and the TGF-β/Cav1 pathway might act as a therapeutic target for hPMSCs in the treatment of LF.

Adeno-associated virus (AAV)-mediated delivery of the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) has shown promising results in preclinical models. However, the long-term expression of Cas9 mediated by AAV in the post-mitotic cells raises concerns with specificity and immunogenicity. Thus, it would be advantageous to limit the duration of Cas9 expression following delivery. In this study, we have engineered an all-in-one self-cleavage AAV-CRISPR-Cas9 system to restrict the expression of Cas9 nuclease, which consists of a Cas9 nuclease from Staphylococcus aureus (SaCas9), a chimeric single guide RNA (sgRNA) molecule targeting PCSK9, and flanking sites targeted by this sgRNA. The self-cleavage system generated a negative feedback loop where Cas9 cut both the target genomic locus and the AAV vector, thus self-limiting the expression of Cas9. We demonstrated that this system could reduce ∼60% expression of SaCas9 protein and had a 20-fold reduction in off-target activity at 24 weeks post-vector administration in vivo. Moreover, the on-target editing efficacy was not compromised and resulted in a stable reduction in circulating PCSK9 and serum cholesterol. The inclusion of this self-cleavage system in gene-editing approaches could increase the safety profile of AAV-delivered genome-editing nucleases and thereby promote its clinical transformation.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-term, stable gene therapy. We have demonstrated SaCas9-mediated homology-directed factor IX (FIX) in situ targeting for sustained treatment of hemophilia B. In this study, we tested a more efficient dual adeno-associated virus (AAV) strategy with lower vector dose for liver-directed genome editing that enables CRISPR-Cas9-mediated site-specific integration of therapeutic transgene within the albumin gene, and we aimed to develop a more universal gene-targeting approach. We successfully achieved coagulation function in newborn and adult hemophilia B mice by a single injection of dual AAV vectors. FIX levels in treated mice persisted even after a two-thirds partial hepatectomy, indicating stable gene integration. Our results suggest that this CRISPR-Cas9-mediated site-specific gene integration in hepatocytes could transform into a common clinical therapeutic method for hemophilia B and other genetic diseases.

Therapeutic antibodies targeting vascular endothelial growth factor (VEGF) have become a critical regimen for tumor therapy, but the efficacy of monotherapy is usually limited by drug resistance and multiple angiogenic mechanisms. Complement proteins are becoming potential candidates for cancer-targeted therapy based on their role in promoting cancer progression and angiogenesis. However, the antitumor abilities of simultaneous VEGF and complement blockade were unknown. We generated a humanized soluble VEGFR-Fc fusion protein (VID) binding VEGFA/PIGF and a CR1-Fc fusion protein (CID) targeting C3b/C4b. Both VID and CID had good affinities to their ligands and showed effective bioactivities. In vitro, angiogenesis effects induced by VEGF and hemolysis induced by complement were inhibited by VID and CID, respectively. Further, VID and CID confer a synergetic therapeutic effect in a colitis-associated colorectal cancer (CAC) model and an orthotopic 4T1 breast cancer model. Mechanically, combination therapy inhibited tumor angiogenesis, cell proliferation, and MDSC infiltration in the tumor microenvironment and promoted tumor cell apoptosis. Our study offers a novel therapeutic strategy for anti-VEGF-resistant tumors and chronic-inflammation-associated tumors. Keywords: VEGFR-Fc fusion protein, CR1-Fc fusion protein, combination therapy, angiogenesis, MDSCs

BackgroundMicrotubule-associated proteins (MAPs) have been considered to play significant roles in the tumor evolution of non-small cell lung cancer (NSCLC). Nevertheless, mRNA transcription levels and prognostic value of distinct MAPs in patients with NSCLC remain to be clarified.MethodsIn this study, the Oncomine database, Gene Expression Profiling Interactive Analysis (GEPIA) database, and Human Protein Atlas were utilized to analyze the relationship between mRNA/protein expression of different MAPs and clinical characteristics in NSCLC patients, including tumor type and pathological stage. The correlation between the transcription level of MAPs and overall survival (OS) of NSCLC patients was analyzed by Kaplan–Meier plotter. Besides, 50 frequently altered neighbor genes of the MAPs were screened out, and a network has been constructed via the cBioPortal and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) dataset. Meanwhile, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on the expression data of MAPs and their 50 frequently altered neighbor genes in NSCLC tissues. Furthermore, The Cancer Immunome Atlas (TCIA) was utilized to analyze the relationship between MAP expression and the response to immunotherapy. Finally, we used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to verify the expression of MAPs in 20 patients with NSCLC.ResultsThe present study discovered that the mRNA transcription levels of MAP7/7D2 were enriched in NSCLC tissues, while those of the MAP2/4/6/7D3 were lower in NSCLC specimens than those in control specimens. The mRNA transcription level of MAP6 was significantly associated with the advanced stage of NSCLC. Besides, survival analysis indicated that higher mRNA expressions of MAP2/4/6/7/7D3 were correlated considerably with favorable OS of NSCLC patients, whereas increased mRNA expression levels of MAP1A/1S were associated with poor OS. Moreover, the expression of MAP1A/1B/1S/4/6/7D1/7D3 was significantly correlated with immunophenoscore (IPS) in NSCLC patients.ConclusionsOur analysis indicated that MAP1A/1S could serve as potential personalized therapeutic targets for patients with NSCLC, and the enriched MAP2/4/6/7/7D3 expression could serve as a biomarker for favorable prognosis in NSCLC. Besides, the expression of MAP1A/1B/1S/4/6/7D1/7D3 was closely related to the response to immunotherapy. Taken together, MAP expression has potential application value in the clinical treatment and prognosis assessment of NSCLC patients, and further verifiable experiments can be conducted to verify our results.

Tumor metastasis is the major cause of poor prognosis and mortality in colorectal cancer (CRC). However, early diagnosis of highly metastatic CRC is currently difficult. In the present study, we screened for a novel biomarker, GDNF family receptor alpha 1 (GFRA1) based on the expression and methylation data in CRC patients from The Cancer Genome Altlas (TCGA), followed by further analysis of the correlation between the GFRA1 expression, methylation, and prognosis of patients. Our results show DNA hypomethylation-mediated upregulation of GFRA1 in invasive CRC, and it was found to be correlated with poor prognosis of CRC patients. Furthermore, GFRA1 methylation-modified sequences were found to have potential as methylation diagnostic markers of highly metastatic CRC. The targeted demethylation of GFRA1 by dCas9-TET1CD and gRNA promoted CRC metastasis in vivo and in vitro. Mechanistically, demethylation of GFRA1 induces epithelial-mesenchymal transition (EMT) by promoting AKT phosphorylation and increasing c-Jun expression in CRC cells. Collectively, our findings indicate that GFRA1 hypomethylation can promote CRC invasion via inducing EMT, and thus, GFRA1 methylation can be used as a biomarker for the early diagnosis of highly metastasis CRC.

Abstract Background Ischemic vascular diseases are the major cause of death worldwide. In recent years, endothelial cell (EC)-based approaches to vascular regeneration are increasingly viable strategies for treating ischemic diseases, but their applications are challenged by the difficulties in their efficient generation and stable maintenance. Here, we show an alternative protocol that facilitates the generation of functional and expandable ETS variant 2 (ETV2)-induced endothelial-like cells (EiECs) from human adipose-derived stem cells (hADSCs), providing a potential source of cells for autologous ECs to treat ischemic vascular diseases. Methods hADSCs were obtained from fresh human adipose tissue. Passage 3 hADSCs were transduced with doxycycline (DOX)-inducible ETV2 transcription factor; purified ETV2-hADSCs were induced into endothelial-like cells using a two-stage induction culture system composed of small molecule compounds and cell factors. EiECs were evaluated for their surface markers, proliferation, gene expression, secretory capacity, and effects on vascular regeneration in vivo. Results We found that short-term ETV2 expression combined with TGF-β inhibition is sufficient for the generation of kinase insert domain receptor (KDR)+ cells from hADSCs within 10 days. KDR+ cells showed immature endothelial characteristics, and they can gradually mature in a chemically defined induction medium at the second stage of induction. Futher studies showed that KDR+ cells deriving EC-like cells could stably self-renew and expand about 106-fold in 1 month, and they exhibited expected genome-wide molecular features of mature ECs. Functionally, these EC-like cells significantly promoted revascularization in a hind limb ischemic model. Conclusions We isolated highly purified hADSCs and effectively converted them into functional and expandable endothelial-like cells. Thus, the study may provide an alternative strategy to obtain functional EC-like cells with potential for biomedical and pharmaceutical applications.

Abstract SARI, also called as BATF2, belongs to the BATF family and has been implicated in cancer cell growth inhibition. However, the role and mechanism of SARI in tumour angiogenesis are elusive. Here we demonstrate that SARI deficiency facilitates AOM/DSS-induced colonic tumorigenesis in mice. We show that SARI is a novel inhibitor of colon tumour growth and angiogenesis in mice. Antibody array and HUVEC-related assays indicate that VEGF has an essential role in SARI-controlled inhibition of angiogenesis. Furthermore, Co-IP/PAGE/mass spectrometry indicates that SARI directly targets ceruloplasmin (Cp), and induces protease degradation of Cp, thereby inhibiting the activity of the HIF-1α/VEGF axis. Tissue microarray results indicate that SARI expression inversely correlates with poor clinical outcomes in colon cancer patients. Collectively, our results indicate that SARI is a potential target for therapy by inhibiting angiogenesis through the reduction of VEGF expression and is a prognostic indicator for patients with colon cancer.

Following publication of the original article [1], the authors reported an error in Fig 5 of this article, graphs presenting FCM and immunofluorescent for CD4T, CD8T and NK cell of the Control Groups (LL2, LL2-irradation, MCS-irradiation) were inadvertently duplicated from another parallel experiment.

Abstract Background Although the whole tumor cell vaccine can provide the best source of immunizing antigens, there is still a limitation that most tumors are not naturally immunogenic. Tumor cells genetically modified to secrete immune activating cytokines have been proved to be more immunogenic. IL-18 could augment proliferation of T cells and cytotoxicity of NK cells. GM-CSF could stimulate dendritic cells, macrophages and enhance presentation of tumor antigens. In our study, we used mouse GM-CSF combined with IL-18 to modify Lewis lung cancer LL/2, then investigated whether vaccination could suppress tumor growth and promote survival. Methods The Lewis lung cancer LL/2 was transfected with co-expressing mouse GM-CSF and IL-18 plasmid by cationic liposome, then irradiated with a sublethal dose X ray (100 Gy) to prepare vaccines. Mice were subcutaneously immunized with this inactivated vaccine and then inoculated with autologous LL/2 to estimate the antitumor efficacy. Results The studies reported here showed that LL/2 tumor cell vaccine modified by a co-expressing mouse GM-CSF and IL-18 plasmid could significantly inhibit tumor growth and increased survival of the mice bearing LL/2 tumor whether prophylactic or adoptive immunotherapy in vivo. A significant reduction of proliferation and increase of apoptosis were also observed in the tumor treated with vaccine of co-expressing GM-CSF and IL-18. The potent antitumor effect correlated with higher secretion levels of pro-inflammatory cytokines such as IL-18, GM-CSF, interferon-γ in serum, the proliferation of CD4+ IFN-γ+, CD8+ IFN-γ+ T lymphocytes in spleen and the infiltration of CD4+, CD8+ T in tumor. Furthermore, the mechanism of tumor-specific immune response was further proved by 51Cr cytotoxicity assay in vitro and depletion of CD4, CD8, NK immune cell subsets in vivo. The results suggested that the antitumor mechanism was mainly depended on CD4+, CD8+ T lymphocytes. Conclusions These results provide a new insight into therapeutic mechanisms of IL-18 plus GM-CSF modified tumor cell vaccine and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.